PROJECT SUMMARY Regulation of elongation by RNA Polymerase II (Pol II) is less well understood than initiation and has wide- ranging importance for gene expression and chromatin structure. Pol II elongation in eukaryotes has several main functions. Of primary importance, Pol II is responsible for expression of protein-coding gene and certain essential snRNA genes. Pol II transcription, including the elongation phase, has wide-ranging impacts on chromatin structure and composition due to cotranscriptional chromatin remodeling and modification. A low level of transcription is pervasive throughout eukaryotic genomes, revealing new challenges to the regulation and control of proper gene expression. A large number of factors have been linked to Pol II transcription during the elongation phase, but their roles in the maintenance and control of Pol II elongation are relatively unknown. The proposed research will identify the mechanisms and regulation of Pol II elongation in vivo using the structurally, genetically and molecularly amenable Saccharomyces cerevisiae. The research builds on the previous identification of a critical role for the highly conserved, conformationally flexible trigger loop (TL) of the Pol II large subunit, Rpb1, in substrate selection, control of elongation rate and as the direct target for the Pol II-elongation specific inhibitor, 1-amanitin. We have generated a series of Pol II mutants with alterations in just a few trigger loop residues that have differences in elongation rate over two orders of magnitude in vitro. In the current work, these mutants with a range of Pol II rates will be used to dissect mechanisms of Pol II elongation in vivo using a multipronged approach. First, it will be determined how mutations that alter Pol II elongation rate in vitro affect Pol II processivity and elongation rate in vivo using chromatin IP, detection of nascent RNA and transcription run on. Second, the consequences of alteration of Pol II activity by substrate limitation in vivo will be determined using a novel genetic system. This system will allow testing of a long standing but unproven phenotype that has been used for the putative identification of elongation factors. Third, a specific model will be tested for Pol II elongation contribution to initiation through the efficiency of promoter escape, wherein transcription begins but may be unsuccessful in reaching a mature elongation phase. This model has broad implications for how elongation factors might control transcription, and how promiscuous initiation may be normally limited. Finally we will determine at nucleotide resolution effects on Pol II pausing, distribution, transcription start site selection and transcriptome content in response to alteration of Pol II TL function. These experiments will reveal the nature of elongation defects in vivo, and how alteration of elongation contributes to gene expression. This work will establish new paradigms for the dissection of Pol II elongation factor function.